Method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same

ABSTRACT

A method of a pretreatment of a sample for quantitating cholesterol characterized by, before measuring cholesterol contained in specific lipoproteins, treating the sample containing lipoproteins with an enzyme, the substrate of which is free cholesterol, optionally together with a reaction accelerator; a method for quantitating cholesterol in specific lipoproteins by using the above method; and a kit for quantitating cholesterol in specific lipoproteins to be used in the above quantification method. By using this quantification method, cholesterol in a specific fraction can be conveniently, accurately and efficiently quantitated fundamentally without resort to polyanion, etc. Thus, this method is appropriately usable in various automatic analyzers.

TECHNICAL FIELD

This invention relates to a pretreatment method for accurately andefficiently discriminating and quantitating cholesterol, which exists inthe specific lipoprotein fraction, by simple procedures while using asmall amount of a sample, and also to a method for measuring cholesterolin the specific lipoprotein fraction by using the pretreatment method.

BACKGROUND ART

Lipids such as cholesterol are complexed with apoproteins in blood toform lipoproteins. Depending on differences in physical properties,lipoproteins are classified into chylomicron, very low densitylipoprotein (VLDL), low density lipoprotein (LDL), high densitylipoprotein (HDL), and so on. Among these lipoproteins, LDL is known tobe one of causative substances which induce arteriosclerosis, while HDLis known to show anti-arteriosclerotic activity.

Epidemiologically, the level of cholesterol in LDL is known to exhibit apositive correlation with the frequency of onset of arterioscleroticdisease while the level of cholesterol in HDL is known to show aninverse correlation with the frequency of onset of arterioscleroticdisease. These days, measurements of cholesterol in LDL or HDL are,therefore, widely conducted for the prevention or diagnosis of ischemicheart diseases.

As methods known for the measurement of cholesterol in LDL or HDL, thereare, for example, a method in which LDL or HDL is separated from otherlipoproteins by ultracentrifugal separation and is then subjected to acholesterol measurement; and another method in which subsequent toseparation of LDL or HDL from other lipoproteins by electrophoresis, itslipid is stained, and the intensity of a developed color is measured.These methods are however not used practically, because they involve oneor more problems in that procedures are intricate and many samplescannot be handled.

A method for the measurement of cholesterol in HDL, which is used atpresent in the field of clinical tests, is the precipitation method inwhich a precipitation reagent is added to a sample to agglutinatelipoproteins other than HDL, the resulting agglutinate is removed bycentrifugation, and cholesterol in isolated supernatant which containsonly HDL is then measured. This method is simpler compared withultracentrifugation or electrophoresis, but due to the inclusion of theprocedures to add the precipitation reagent and to perform theseparation, requires each sample is a relatively large quantity, andinvolves a potential problem of causing an analytical error.Furthermore, the entire analysis steps of this method can not be fullyautomated.

On the other hand, enzymatic methods have been studied for thefractional quantitation of cholesterol in HDL. Known methods include,for example, to conduct an enzymatic reaction in the presence of a bileacid salt and a nonionic surfactant (JP 63-126498 A). This method makesuse of the fact that an enzymatic reaction proceeds in proportion to theconcentration of cholesterol in LDL in an initial stage of the reactionand the subsequent reaction velocity is in proportion to theconcentration of cholesterol in HDL. A problem however exists inaccuracy because the reaction with the cholesterol in HDL and thereaction with cholesterol in other lipoproteins cannot be fullydistinguished.

Also included in the known methods is to have lipoproteins other thanHDL agglutinated in advance, to cause cholesterol in HDL alone to reactenzymatically, and to inactivate the enzyme and at the same time, toredissolve the agglutinate, followed by the measurement of an absorbance(JP 6-242110 A). This method, however, requires at least threeprocedures to add reagents so that it can be applied only to particularautomated analyzers, leading to a problem in a wide applicability.Further, this method is not satisfactory from the standpoint of damagesto analytical equipment and disposal of the reagents because of the useof a salt at a high concentration upon redissolution of an agglutinate.

A still further method is also known (JP 9-299 A), which comprisescausing, in a first reaction, cholesterol oxidase and cholesterolesterase to act upon lipoproteins other than HDL in the presence of aspecial surfactant and to have cholesterol, which is contained in suchother lipoproteins, preferentially reacted, and then measuringcholesterol in HDL while inhibiting any reaction to cholesterol inlipoproteins other than HDL. This method, however, is considerablydifferent from the present invention inter alia in that in the firstreaction, the special surfactant, cholesterol oxidase and cholesterolesterase are required at the same time to put, outside the reactionsystem, both free cholesterol and esterified cholesterol in thelipoproteins other than HDL.

Further, Japanese Patent No.2,600,065 discloses a method which makescombined use of a precipitation reagent, which is adapted to causeprecipitation of lipoproteins other than HDL, and a cholesterolmeasuring reagent to measure cholesterol (HDL-C) in unprecipitated HDL.This method has practical utility when a modified enzyme is used asenzyme and α-cyclodextrin sulfate is used as a precipitation reagent.This method, however, also involves a problem in accuracy in thatturbidity, which occurs as a result of the use of the precipitationreagent, interferes with the measurement system.

Concerning the measurement of HDL-C by a modified enzyme, “SEIBUTSUSHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)”, 19(5), 305-320, whichis considered to be a published paper on the above-described patentedmethod, discloses that, under the recognition of incapability ofmeasurement of HDL-C in a serum of a hyperlipidemic patient by themodified enzyme due to a positive error (that is, to result in a highervalue compared with that obtained by the precipitation method) inducedwhen the modified enzyme is simply introduced into a reaction system,HDL-C was measured by using cyclodextrin sulfate, a polyanion, andmagnesium chloride as a precipitation reagent for the avoidance of thepositive error.

To reduce the influence of turbidity caused by a precipitation reagentin the above-described patented method, certain techniques are alsoknown, including to make a surfactant exist concurrently (JP 8-116996A), to use an antibody (JP 9-96637 A), and to employ a sugar compound(JP 7-301636 A). They, however, all require as a premise the inclusionof a reagent which induces formation of an agglutinate, so that it isfundamentally indispensable for them to use a precipitation reagent suchas a polyanion.

The present inventors recently found that use of a substance, which actsupon the specific lipoprotein only, makes it possible to accuratelyquantitate cholesterol in the specific lipoprotein fraction withoutusing a precipitation reagent, and filed patent applications (JP9-244821). This method has an extremely high correlation with theconventional precipitation method, but compared in measurement valueswith the precipitation method, this method is recognized to have asimilar tendency as the above-described method reported in “SEIBUTSUSHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)”. To obtain dataconsistent with those obtained by the conventional precipitation methodat medical institutions and the like, a polyanion or the like is added.

From the standpoint of the problem of a tarnish or the like on a cuvetteand scattering of measurement values, however, it is not desired to adda polyanion or the like and to form a precipitate in a measurementsystem. Accordingly, it has been strongly desired to eliminate theprecipitate from the system. Further, it is also economicallyunreasonable to use a polyanion or the like for making the resultingdata consistent with those obtained by the precipitation method althoughthe polyanion or the like is not needed from the standpoint of theprinciple of the measurement. Hence, there is also an outstanding desirefor its solution.

An object of the present invention is, therefore, to provide a method,which can accurately and efficiently quantitate cholesterol in thespecific lipoprotein fraction by simple procedures fundamentally withoutneeding a polyanion or the like and is suitably applicable to variousautomated analyzers.

DISCLOSURE OF THE INVENTION

The present inventors proceeded with a thorough investigation for acause which may be responsible for the above-described problem reportedin “SEIBUTSU SHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)”, that is,the problem that a value of cholesterol in the specific lipoproteinfraction as quantitated by using a substance which acts only upon aspecific lipoprotein such as HDL becomes higher than the correspondingvalue as determined by the precipitation method; and came to aconclusion that even from non-HDL lipoproteins (LDL, VLDL and the like)the cholesterol of which is not supposed to be measured, a small amountof free cholesterol existing on their surfaces or in the vicinity oftheir surfaces is liberated to cause a positive error. Based on thisfinding, it has been found that a cholesterol value obtained by aquantitation method making use of a substance, which acts upon aspecific lipoprotein only, becomes consistent with the correspondingvalue obtained by the precipitation method when the cholesterol value ismeasured after consuming only free cholesterol in advance underconditions that lipoproteins remain substantially unchanged, leading tothe completion of the present invention.

Described specifically, the present invention provides a method forpretreating a sample, which contains various lipoproteins, prior tomeasuring cholesterol existing in specific one of the lipoproteins inthe sample, which comprises causing an enzyme, which acts upon freecholesterol as a substrate, to act upon the sample to consume only thefree cholesterol in advance under conditions that the lipoproteinsremain substantially unchanged.

The present invention also provides a method for quantitatingcholesterol existing in a specific lipoprotein in a sample, whichcomprises causing an enzyme, which acts upon free cholesterol as asubstrate, to act upon the sample with the lipoprotein contained thereinto consume only the free cholesterol under conditions that thelipoproteins remain substantially unchanged; and then measuring thecholesterol, which exists in the specific lipoprotein, by using asubstance which acts upon the specific lipoprotein only.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a correlation between the present inventionin Example 1 and the precipitation method;

FIG. 2 is a diagram showing a correlation between the present inventionin Example 2 and the precipitation method; and

FIG. 3 is a diagram showing effects of a reaction accelerator in Example5.

BEST MODES FOR CARRYING OUT THE INVENTION

In the present invention, before measuring cholesterol existing in aspecific lipoprotein in a sample, an enzyme which acts upon freecholesterol as a substrate is caused to act, as pretreatment, upon thesample such that the free cholesterol is consumed.

As the enzyme which acts upon free cholesterol as a substrate, anyenzyme can be used insofar as it acts upon free cholesterol as asubstrate. Illustrative are cholesterol dehydrogenase and cholesteroloxidase. They can be of any origins such as microorganism origins,animal origins or plant origins, and can also be those prepared bygenetic engineering. Further, they can be either modified or unmodifiedchemically. The enzyme is generally used at 0.001 to 100 U/mL, with 0.1to 100 U/mL being preferred.

No particular limitation is imposed on conditions under which theabove-described enzyme, which acts upon free cholesterol as a substrate,is caused to act upon the sample, and conditions recommended for theenzyme can be used. It is however necessary to pay attention so that,during a stage in which the enzyme which acts upon free cholesterol as asubstate is caused to act upon the sample, a reaction through which anesterified cholesterol is converted into free cholesterol does not takeplace. Namely, it is not important whether or not cholesterol esteraseexists. What is needed is to maintain conditions such that cholesterolesterase is not allowed to act practically.

Along with the enzyme which acts upon free cholesterol as a substrate, acoenzyme can be used as needed. As the coenzyme, nicotinamide adeninedinucleotide or the like is usable. Such coenzymes can be used eithersingly or in combination. The amount to be used varies depending on thecoenzyme. The coenzyme may be used at 0.001 to 100 U/mL, preferably at0.1 to 100 U/mL, although no particular limitation is imposed thereon.

Concerning the enzyme which acts upon free cholesterol as a substrateand is used in the present invention, no limitation is imposed on itsorigin as described above. Its concentration and the like can be chosensuitably to achieve desired performance and handling ease. Accordingly,if it is desired to have the pretreatment completed in a predeterminedtime, for example, it is only necessary to use the enzyme in a greateramount, and if it is conversely desired to save the enzyme, it is onlynecessary to make the pretreatment time longer.

In the case of a diagnostic reagent for exclusive use in measurements byautomated analyzers, however, it is desired to meet both of therequirements at the same time. Namely, it is required to complete thepretreatment in a short time by using the enzyme in a small amount. Insuch a case, concurrent existence of a reaction accelerator selectedfrom the below-described group in the pretreatment, which uses an enzymewhich acts upon free cholesterol as a substrate, makes it possible toachieve desired performance with a reduced amount of the enzyme withoutmaking the pretreatment time longer.

Reaction accelerators usable for the above purpose can include, forexample, flufenamic acid, mefenamic acid, 2,2′,6′,2″-terpyridine, tiglicacid, fusidic acid, betamethasone acetate, monensin and mevinolin,including their salts and metal derivatives (aluminum derivatives andthe like) wherever such salts and metal derivatives exist. Among these,flufenamic acid and mefenamic acid are known as non-steroidalanti-inflammatory drugs, and fusidic acid and monensin are known asantibiotics.

Upon using such a compound as a reaction accelerator, it is necessary tosuitably choose its concentration and the like by taking intoconsideration its physical properties, pH and ionic strength of themeasurement system, and the kinds and concentrations of substancesexisting together.

The concentration of the reaction accelerator can be experimentallydetermined in accordance with conditions of a measuring system. Ingeneral, however, flufenamic acid can may be used at about 0.01 to 100mM; fusidic acid at about 0.01 to 10 mM; mefenamic acid,2,2′,6′,2″-terpyridine and betamethasone acetate, each, at about 0.01 to5 mM; monensin and mevinolin, each, at about 0.01 to 1 mM; and tiglicacid at about 1 to 500 mM.

Use of the above-described reaction accelerator has made it possible toreduce the amount of the enzyme, which acts upon free cholesterol as asubstrate, to one severalth or to one several tenth. When the enzyme isused in the same amount, on the other hand, the reaction accelerator canshorten the reaction time.

In the above-described pretreatment by the enzyme which acts upon freecholesterol as a substrate (and also by the reaction accelerator, ifneeded), it is also possible to use other enzymes (with exclusion ofthose giving substantial influence to lipoproteins) and salts, buffersfor pH regulation, surfactants (with exclusion of those givingsubstantial influence to lipoproteins), preservatives, proteins such asalbumin, and agents having affinity to specific lipoproteins, such asantibodies, antibiotics, saponins, lectins and polyanions to extents notcausing agglutination of the specific lipoprotein, such that the actionof the enzyme is adjusted without impairing the specificity of themeasurement.

In the present invention, those containing the following ingredientscan, therefore, be used as pretreatment agents for measuring cholesterolexisting in specific lipoproteins in samples.

(Essential ingredients)

Enzymes which act upon free cholesterol as a substrate, for example,cholesterol dehydrogenase and cholesterol oxidase.

(Optional ingredients)

Reaction accelerators, for example, flufenamic acid, mefenamic acid,2,2′,6′,2″-terpyridine, tiglic acid, fusidic acid, betamethasoneacetate, monensin and mevinolin.

(Other ingredients)

coenzymes such as NAD, other enzymes such as peroxidase, catalase,diaphorase and ascorbate oxidase, acids such as pyruvic acid, salts,buffers for pH regulation, surfactants giving no substantial influenceon lipoproteins, preservatives, proteins such as albumin, antibodies,antibiotics, saponins, lectins, polyanions and couplers such as4-aminoantipyrine, oxidative color developers such as hydrogen donors,e.g., Trinder's reagent, electron acceptors such as phenazinemethosulfate, and reductive color developers such as nitrobluetetrazolium.

In the present invention, cholesterol which exists in a specificlipoprotein in a sample is measured after having free cholesterol inlipoproteins consumed by the above-described pretreatment.

Any method can be used for the measurement of the cholesterol existingin the specific lipoprotein in the sample insofar as the method canmeasure the cholesterol existing in the specific lipoprotein by using asubstance which acts upon the specific lipoprotein only.

An illustrative example of the method may comprise providing, as thesubstance which acts upon the specific lipoprotein, a surfactantselected from polyoxyethylene alkylene phenyl ethers or polyoxyethylenealkylene tribenzylphenyl ethers disclosed in JP 11-56395 A; adding acholesterol measuring enzyme reagent in the presence of the substance;and then measuring the amount of cholesterol reacted in a time duringwhich cholesterol in high density lipoprotein out of lipoproteinspreferentially reacts with the cholesterol measuring enzyme reagent.

Examples of commercial products of the former surfactants,polyoxyethylene alkylene phenyl ethers, can include “Emulgen A-60”(trade name, product of Kao Corporation), while examples of commercialproducts of the latter surfactants, polyoxyethylene alkylenetribenzylphenyl ethers, can include “Emulgen B66” (trade name, productof Kao Corporation).

As an alternative method, there is a method which makes use of themodified enzymes, which are disclosed on pages 305-320 of “SEIBUTSUSHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)”, 19(5), as substanceswhich act only upon specific lipoproteins, respectively. Althoughα-cyclodextrin sulfate and magnesium chloride are used in the method ofthis paper to inhibit reactions with lipoprotein fractions other thanHDL, the use of the above-described pretreatment method of thisinvention makes it no longer necessary to use such substances.

Except for the use of the substance which acts upon the specificcholesterol, the method for the measurement of cholesterol existing inthe specific lipoprotein can be practiced by using reagents employed inconventional cholesterol-measuring methods. Examples of ingredientswhich may be contained in reagents to be used can include enzymes suchas cholesterol esterase, cholesterol oxidase, cholesterol dehydrogenase,isocitrate dehydrogenase, diaphorase and peroxidase, color developers,coenzymes, electron acceptors, proteins (albumin, etc.), preservatives,surfactants, salts, acids, and buffers for pH regulation.

As surfactants out of the above-described ingredients, both ionic andnonionic surfactants are usable. Illustrative are polyoxyethylene alkylethers, polyoxyethylene alkylphenyl ethers,polyoxyethylene-polyoxypropylene condensate, polyoxyethylene alkyl ethersulfates, alkylbenzenesulfonate salts, and bile acid salts. The amountof the surfactant to be used varies depending on the compound. Thesurfactant may however be used in 0.0001% to 5%, preferably in 0.001% to5%, although no particular limitation is imposed thereon.

No particular limitation is imposed on the buffers. Conventional bufferssuch as Good's buffer, phosphate buffer, Tris buffer and phthalatebuffer are usable. The buffer may be used at 0.005 M to 2 M, preferably0.01 M to 1 M, although no particular limitation is imposed thereon.

The method for quantitating cholesterol in a specific lipoproteinfraction by the present invention typically comprises firstly adding apretreatment agent, which acts upon free cholesterol only, into ameasureing sample and causing the pretreatment agent to act upon thesample, and then adding and mixing a cholesterol measuring reagent(hereafter called a “quantitation reagent”), which contains a substancecapable of acting upon the specific lipoprotein and a reagent employedfor a conventional cholesterol-measuring method, to measure the amountof cholesterol in the specific lipoprotein fraction.

Specific examples can include, but are not limited to, a method whichcomprises mixing cholesterol dehydrogenase and a coenzyme (NAD) with asample and then adding a cholesterol-measuring reagent which comprisescholesterol esterase and cholesterol oxidase; a method which comprisesmixing cholesterol dehydrogenase and NAD with a sample and then adding acholesterol-measuring reagent which comprises cholesterol esterase; amethod which comprises mixing a sample and cholesterol oxidase togetherwith peroxidase, 4-amino antipyrine or catalase and then adding acholesterol-measuring reagent which comprises cholesterol esterase; anda method which comprises mixing a sample and cholesterol oxidasetogether with peroxidase, 4-aminoantipyrine, etc. and then adding acholesterol-measuring reagent which comprises cholesterol esterase,cholesterol dehydrogenase and NAD.

Examples of the method for measuring cholesterol in a specificlipoprotein fraction can include a method making combined use ofcholesterol esterase and cholesterol oxidase as an enzyme reagent and amethod making combined use of cholesterol esterase and cholesteroldehydrogenase, although known enzyme assays are all usable.

In the present invention, the enzyme for use in the first reaction asthe pretreatment reaction and the enzyme for use in the measurement ofcholesterol as the quantitation method through the second reaction maybe either the same or different. Further, the enzyme may be used in anexcess amount in the first reaction and may also be used in the secondreaction. In essence, it is only necessary to consume free cholesterol,which exists in a small amount on lipoprotein surfaces, (firstreaction/pretreatment reaction) and then to bring the reaction systeminto a state, in which the enzyme acts only upon the specificlipoprotein to be measured, so that most cholesterol (freecholesterol+esterified cholesterol) forming the lipoprotein can bequantitated.

Further, no particular limitation is imposed on the method for finallydetecting cholesterol after the addition of such a cholesterol-measuringenzyme reagent. It is possible to use, for example, absorptiometry inwhich detection is conducted by combining peroxidase with a chromogen ordiaphorase or an electron acceptor with a reductive color-developingreagent; or a method in which a coenzyme or hydrogen peroxide isdirectly detected. The coenzyme may be amplified by a coenzyme cyclingsystem.

To practice the method of the present invention with ease, it ispreferred to use a quantitation kit which is suited for measuringcholesterol in the specific lipoprotein.

Although such kits can be readily designed based on the aboveexplanation, their examples will be described next by dividing them intothose making use of cholesterol oxidase and those making use ofcholesterol dehydrogenase as typical example of enzymes which act uponfree cholesterol as a substrate.

[Kits Making Use of Cholesterol Oxidase]

(a) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol oxidase and a hydrogenperoxide consuming substance (and further comprising a reactionaccelerator in some instances); and

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only, cholesterol esterase, and a color developer.

(b) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol oxidase, cholesterolesterase, and a hydrogen peroxide consuming substance (and furthercomprising a reaction accelerator in some instances); and

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only, and a color developer.

(c) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1), (2) and (3):

(1) a first reagent comprising cholesterol oxidase and a hydrogenperoxide consuming substance (and further comprising a reactionaccelerator in some instances);

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only; and

(3) a third reagent comprising cholesterol esterase and a colordeveloper.

In the above-described kits, the term “hydrogen peroxide consumingsubstance” means a substance which consumes and eliminates hydrogenperoxide produced by the reaction between cholesterol oxidase andcholesterol. Illustrative are catalase, couplers such as4-aminoantipyrine, and oxidative-reductive color developer agentsincluding hydrogen donors such as Trinder's reagent.

Among these, a coupler such as 4-aminoantipyrine and a hydrogen donorsuch as Trinder's reagent develop a color when reacted, in combination,with hydrogen peroxide, and are usable as the color developer in theabove-described reagent (2) or (3). As the reagent (1) for use in thepretreatment step according to the present invention, it is preferred touse only one of a coupler and a hydrogen donor and to have hydrogenperoxide consumed through a non-color developing reaction. Needless tosay, it is also possible to subject hydrogen peroxide to acolor-developing reaction and then to make an adjustment to a measuredvalue [this adjustment can be made by subtracting the intensity of acolor, which is developed by the reagent (1), from the intensity of acolor developed by the reagent (2) or the reagent (3)].

[Kits Making Use of Cholesterol Dehydrogenase]

(d) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol dehydrogenase and acoenzyme(and further comprising a reaction accelerator in someinstances); and

(2) a second reagent comprising a substance, which acts upon thespecific lipoprotein only, and cholesterol esterase.

(e) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol dehydrogenase and acoenzyme(and further comprising a reaction accelerator in someinstances); and

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only, cholesterol oxidase, cholesterol esterase, peroxidase,and a color developer.

(f) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol dehydrogenase, a coenzyme,and cholesterol esterase(and further comprising a reaction acceleratorin some instances); and

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only.

(g) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol dehydrogenase, a coenzyme,and cholesterol esterase(and further comprising a reaction acceleratorin some instances); and

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only, cholesterol oxidase, peroxidase, and a colordeveloper.

(h) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1), (2) and (3):

(1) a first reagent comprising cholesterol dehydrogenase and acoenzyme(and further comprising a reaction accelerator in someinstances);

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only; and

(3) a third reagent comprising cholesterol esterase.

(i) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1), (2) and (3):

(1) a first reagent comprising cholesterol dehydrogenase and acoenzyme(and further comprising a reaction accelerator in someinstances);

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only; and

(3) a third reagent comprising cholesterol oxidase, cholesterolesterase, peroxidase, and a color developer.

(j) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol dehydrogenase, a coenzyme,and a coenzyme reaction product consuming substance (and furthercomprising a reaction accelerator in some instances); and

(2) a second reagent comprising a substance, which acts upon thespecific lipoprotein only, and cholesterol esterase.

(k) A quantitation kit for cholesterol in a specific lipoprotein,comprising the following reagents (1) and (2):

(1) a first reagent comprising cholesterol dehydrogenase, a coenzyme,and a coenzyme reaction product consuming substance(and furthercomprising a reaction accelerator in some instances); and

(2) a second reagent comprising a substance which acts upon the specificlipoprotein only, cholesterol esterase, and a color developer.

In the above-described kits making use of cholesterol dehydrogenase, theterm “coenzyme reaction product consuming substance” means a substancewhich converts a reduced coenzyme (for example, NADH), which occursthrough the reaction among cholesterol, cholesterol dehydrogenase and acoenzyme (for example, NAD), back into the original coenzyme.Illustrative is a combination of lactate dehydrogenase and pyruvic acid(substrate). In each of the above-described kits, the reaction productof the coenzyme is produced by the addition of the reagent (1). In eachof the kits (d), (f), (h) and (j) out of the above-described kits, lightof the same wavelength as a color developed by the addition of thereagent (1) may be measured in the measurement stage without advanceconsumption of the reaction product. In this case, however, it isnecessary to quantitate the cholesterol in the specific lipoprotein bysubtracting the intensity of a color, which is developed in thepretreatment stage in which the reagent (1) is added, from the intensityof a color developed by the reagent (2) or the reagent (3). As analternative, it may also be possible to add beforehand the substance,which consumes the reaction product, to the reagent (1) and subsequentto consumption of the reaction product, to add the reagent (2) or thereagent (3) for the development of a color. In this case, addition of asubstance, which reduces the action of the substance which consumes thereaction product, to the reagent (2) or the reagent (3) is preferred. Ineach of the kits (e), (g), (I) and (k), on the other hand, it is notabsolutely necessary to subtract the intensity of the color, which isdeveloped in the pretreatment stage, from the color intensity measuredin the measurement stage, because in the measurement stage, a developedcolor of a wavelength different from the color developed in thepretreatment stage is measured.

It is to be noted that the application of the above-mentioned reactionaccelerators, such as flufenamic acid, mefenamic acid,2,2′,6′,2″-terpyridine, tiglic acid, fusidic acid, betamethasoneacetate, monensin and mevinolin, is limited neither to the pretreatmentmethod or agent of the present invention nor the quantitation method orkit of the present invention for cholesterol in a specific lipoprotein,said quantitation method or kit making use of the pretreatment method oragent.

If a reaction accelerator such as fulfenamic acid is allowed to existconcurrently upon conducting a cholesterol quantitation method makinguse of an enzyme which acts upon free cholesterol as a substrate, forexample, a free cholesterol quantitation method making combined use ofcholesterol oxidase, peroxidase, a color developer and the like or atotal cholesterol quantitation method making combined use of cholesteroloxidase, cholesterol esterase, peroxidase, a color developer and thelike, it is obviously possible to bring about advantageous effects suchthat the amount of the enzyme to be used, said enzyme being capable ofacting upon free cholesterol as a substrate and being cholesteroloxidase in the above-exemplified method, can be reduced and the time ofthe enzymatic reaction can be shortened.

Further, reference to the disclosure of this specification on thecholesterol quantitation method (for example, selection of a surfactantto limit a target of a specific lipoprotein to be measured) makes itpossible to more specifically design a quantitation method as desired.

INDUSTRIALLY APPLICABILITY

The present invention has made it possible to efficiently quantitatecholesterol in a specific fraction by simple procedures without using apolyanion or the like, to say nothing of a mechanical pretreatment suchas centrifugation. As the methods of the present invention do not form aprecipitate which would otherwise occur by the addition of the polyanionor the like, measuring apparatus (especially, cuvettes) and the likeremain free of a tarnish and moreover, measured values also remain freeof scattering. The methods according to the present invention are,therefore, superior to the conventional cholesterol measuring methods.

Further, as will be demonstrated in subsequent Examples, measurementvalues showing a high correlation with those obtained by theconventional precipitation method can be obtained even with respect tosamples with high triglyceride levels. Therefore, the methods accordingto the present invention are also excellent in that they are applicableto various samples without limitation.

In addition, the use of the reaction accelerator makes it possible touse the enzyme, which acts upon free cholesterol as a substrate, in asmaller amount in the pretreatment stage.

As has been described above, the methods according to the presentinvention permit accurate and specific measurements of a variety ofsamples by simple procedures while using the samples in smallquantities. Accordingly, they can be applied to various automatedanalyzers and are also extremely useful in the field of clinical tests.

The present invention will next be described in further detail by theExamples. It should however be borne in mind that the present inventionis by no means limited to the Examples.

EXAMPLE 1

With respect to each of 30 serum samples containing lipoproteins, thecholesterol in HDL was quantitated by the below-described methodaccording to the present invention and the precipitation method, and themeasurement values were compared.

(Invention Method)

10 mM phosphate buffer (First Reagent; pH 8.5) (300 μL), which contained0.1 U/mL cholesterol dehydrogenase (product of Amano Pharmaceutical Co.,Ltd.), 2.5 mM NAD and 0.03% 4-aminoantipyrine, was added to each sample(3 μL) (pretreatment). About 5 minutes later, a cholesterol quantitationreagent (Second Reagent) (100 μL)—which was composed of 100 mM MESbuffer (pH6) containing 1% “Emulgen B-66”, 1.3 U/mL cholesterol esterase(product of Asahi Chemical Industry Co., Ltd.), 2 U/mL cholesteroloxidase (product of Asahi Chemical Industry Co., Ltd.), 5 U/mLperoxidase (product of Toyobo Co., Ltd.) and 0.04%disulfobutylmetatoluidine—was added.

Just before the addition of the Second Reagent and upon an elapsed timeof five minutes after the addition, the absorbance was measured at 600nm. From a difference in absorbance, the concentration of HDLcholesterol in the serum sample was determined (2-point method). As acalibration substance, a control serum sample with a known concentrationof HDL cholesterol was used. The above procedures were conducted using“Hitachi 7150 automated analyzer”.

(Precipitation Method)

“HDLC 2 ‘Daiichi’ Precipitant” (product of Daiichi Pure Chemicals Co.,Ltd.) (200 μL) was mixed with the sample (200 μL), followed bycentrifugation at 3,000 rpm for 10 minutes. The supernatant (50 μL) wascollected, followed by the mixing with a cholesterol quantitationreagent (3 mL) composed of 100 mM MES buffer (pH6.5) containing 1%Triton X-100, 1 U/mL cholesterol esterase, 1 U/mL cholesterol oxidase, 5U/mL peroxidase, 0.04% disulfobutylmetatoluidine and 0.04%4-aminoantipyrine. After the resulting mixture was incubated at 37° C.for 10 minutes, its absorbance at 600 nm was measured to determine theconcentration of the cholesterol in HDL.

(Results)

The results are shown in Table 1 and FIG. 1.

TABLE 1 Precipitation Invention Sample No. method (mg/dL) method (mg/dL)1 73 72 2 39 39 3 53 52 4 54 54 5 57 58 6 75 71 7 51 51 8 52 50 9 43 4310 58 58 11 59 59 12 49 51 13 44 46 14 70 65 15 35 38 16 54 54 17 45 4718 60 59 19 50 52 20 58 56 21 38 39 22 56 55 23 35 37 24 29 31 25 63 6026 51 50 27 33 36 28 52 51 29 65 63 30 47 49

As is readily envisaged from the results, the invention method, despitethe omission of a polyanion or the like, showed an extremely goodcorrelation with the conventional precipitation method.

EXAMPLE 2

Measurements were conducted by another method of the present invention,which was similar to the invention method conducted in Example 1 exceptthat in the first reagent, cholesterol dehydrogenase, NAD and thephosphate buffer were replaced by 5 U/mL cholesterol oxidase (product ofToyobo Co., Ltd.), 5 U/mL peroxidase (product of Toyobo Co., Ltd.) and100 mM MES buffer (pH 6). The measurement values were compared withthose obtained by the precipitation method in Example 1.

(Results)

The results are shown in Table 2 and FIG. 2.

TABLE 2 Precipitation Invention Sample No. method (mg/dL) method (mg/dL)1 73 73 2 39 38 3 53 52 4 54 56 5 57 57 6 75 74 7 51 52 8 52 50 9 43 4410 58 58 11 59 57 12 49 51 13 44 45 14 70 69 15 35 37 16 54 54 17 45 4718 60 61 19 50 50 20 58 55 21 38 37 22 56 56 23 35 35 24 29 29 25 63 6126 51 52 27 33 33 28 52 52 29 65 66 30 47 46

As is readily envisaged from the results, the invention method, despitethe omission of a polyanion or the like, showed an extremely goodcorrelation with the conventional precipitation method.

EXAMPLE 3

Using the reagents of Example 1 and Example 2, five serum samples ofdifferent triglyceride levels were measured. The measurement values werethen compared with those obtained by the precipitation method. Theresults are shown in Table 3.

TABLE 3 Invention Invention Precipitation method in method inTriglyceride method Example 1 Example 2 level (mg/dL) (mg/dL) (mg/dL)(mg/dL) Sample A 47 49 49 198 Sample B 49 50 49 301 Sample C 26 27 24742 Sample D 60 61 61 517 Sample E 37 40 36 428

As is shown in Table 3, measurement values of comparable levels withthose obtained by the conventional method were also obtained by thepresent invention with respect to the samples of the high triglyceridelevels.

EXAMPLE 4

Measurements were conducted in a similar manner as in Example 2 exceptthat in the first reagent, 5 U/mL cholesterol oxidase was changed togive reagent compositions of the ingredient concentrations andcombinations shown below in Table 4. The measurement values werecompared with those obtained by the precipitation method and also withthose obtained by the invention method (standard test system) of Example2. Incidentally, as a second reagent, the same reagent as the secondreagent employed in Example 1 was used. The results are shown in Table5.

(Compositions of Testing Reagents)

TABLE 4 Test system Contents of composition Standard Cholesterol oxidase(5 U/mL) A Cholesterol oxidase (1 U/mL) B Flufenamic acid + cholesteroloxidase (0.15 mM)    (1 U/mL) C Mefenamic acid + cholesterol oxidase(0.1 mM)    (1 U/mL) D 2,2′,6′,2″-terpyridine + cholesterol oxidase (0.5mM)    (1 U/mL) E Tiglic acid + cholesterol oxidase (50 mM)     (1 U/mL)F Fusidic acid + cholesterol oxidase (0.1 mM)    (1 U/mL) GBetamethasone acetate + cholesterol oxidase (0.2 mM)    (1 U/mL) HMonensin + cholesterol oxidase (0.2 mM)    (1 U/mL) I Mevinolin +cholesterol oxidase (0.05 mM)    (1 U/mL)

(Results)

TABLE 5 Precipitation method Test system (mg/dL) Sample (mg/dL) StandardA B C D E F G H I  1 80 77 72 77 68 76 74 74 74 74 76  2 76 74 72 74 6473 71 73 74 74 73  3 75 72 70 72 66 71 70 70 70 71 71  4 71 72 71 70 6669 69 71 69 71 72  5 71 70 70 69 61 68 67 68 70 69 70  6 71 70 67 70 6368 68 67 69 70 68  7 69 66 63 66 61 65 65 65 64 65 66  8 67 69 70 68 6068 67 66 69 68 68  9 66 65 65 65 59 65 64 63 65 65 65 10 65 65 64 65 5865 64 62 64 65 63 11 57 58 56 57 54 57 56 57 57 58 57 12 56 56 55 55 4955 54 53 55 55 55 13 54 55 54 55 50 54 53 53 53 54 54 14 53 54 54 52 4653 52 52 54 52 53 15 52 53 52 51 47 52 51 49 52 51 52 16 51 53 51 50 4650 51 49 51 51 51 17 49 50 48 48 44 47 48 47 48 48 49 18 47 48 48 46 4146 46 45 47 47 47 19 45 46 48 44 38 46 43 45 47 47 46 20 47 47 49 45 4046 45 45 48 47 47 21 42 44 44 43 39 43 42 41 43 44 43 22 39 42 43 41 3741 41 39 41 41 41 23 32 35 36 33 31 36 34 32 34 34 33 24 18 20 22 19 1723 19 18 21 20 19 25 40 42 42 41 38 45 41 40 41 42 41 Correlation coef.— 0.996 0.990 0.998 0.992 0.995 0.997 0.997 0.994 0.995 0.997 Slope0.905 0.838 0.941 0.832 0.856 0.888 0.915 0.877 0.891 0.917 Intercept5.6 8.7 2.6 3.4 7.5 4.6 2.8 6.3 5.7 4.1

When the amount of cholesterol oxidase was reduced to one fifth (thetest system A) compared with the standard test system (Example 2), thecorrelation coefficient slightly declined and the value of interceptslightly increased. When the reaction accelerator was used, however,results substantially comparable with those of the standard test systemwere obtained even when the amount of cholesterol oxidase was one fifth.It has hence become evident from these results that the use of areaction accelerator makes it possible to reduce the amount ofcholesterol oxidase to be used.

EXAMPLE 5

Reagents J to L shown below in Table 6 were prepared, which commonlycontained 1.25 U/mL peroxidase (product of Toyobo Co., Ltd.), 0.01%4-aminoantipyrine, 0.02% disulfo butyl-m-toluidine and 50 mM NaCl andwere different from each other in the kind and pH of buffer and theconcentrations of cholesterol oxidase (product of Toyobo Co., Ltd.) andfulfenamic acid (product of Sigma Chemical Co.).

TABLE 6 {circle around (1)} Buffer (pH) {circle around (2)}Concentration of cholesterol oxidase Reagent {circle around (3)}Concentration of fulfenamic acid J {circle around (1)} 50 mM Bis-Tris(pH 6.0) {circle around (2)} 0.5, 1.0, 2.5, 5.0 U/mL {circle around (3)}0, 0.01, 0.05, 0.1 mM K {circle around (1)} 50 mM PIPES (pH 7.0) {circlearound (2)} 0.5, 1.0, 2.5, 5.0 U/mL {circle around (3)} 0, 0.1, 0.5, 1.0mM L {circle around (1)} 50 mM MOPS (pH 8.0) {circle around (2)} 0.5,1.0, 2.5, 5.0 U/mL {circle around (3)} 0, 1.0, 5.0, 10.0 mM

Reagents J to L (300 μL) were separately added to aliquots (3 μL) ofeach serum sample. After the resultant mixtures were incubated at 37° C.for 5 minutes, their absorbances were measured at600 nm. The aboveprocedures were conducted using the Hitachi 7150 automated analyzer.

Four serum samples were measured with Reagents J to L.

With respect to each of Reagents J to L, relative absorbances werecalculated for the individual concentrations of cholesterol oxidase andfulfenamic acid by assuming that the absorbance obtained with a reagentcontaining 5.0 U/mL cholesterol oxidase and 0 mM fulfenamic acid was100.

(Results)

Results, which had been obtained by averaging the relative absorbancesof the four samples, are presented in FIG. 3. in which “COD” stands forcholesterol oxidase.

As is readily appreciated from the results, the relative absorbanceincreased depending upon the concentration of fulfenamic acidirrespective of the pH. It has, therefore, been confirmed that the useof the reaction accelerator makes it possible to reduce the amount ofcholesterol oxidase to be used.

It has also become clear that the reaction accelerator is also usable ina method for the measurement of free cholesterol or total cholesterol,which makes use of an enzyme which acts upon free cholesterol as asubstrate.

What is claimed is:
 1. A method for pretreating a sample, which containsvarious lipoproteins, prior to measuring cholesterol existing inspecific one of said lipoproteins in said sample, which comprisescausing an enzyme, which acts upon free cholesterol as a substrate, anda reaction accelerator, which is selected from flufenamic acid,mefenamic acid, 2,2′,6′,2″-terpyridine, tiglic acid, fusidic acid,betamethasone acetate, monensin and mevinolin, to act upon said sampleto convert only said free cholesterol in advance under conditions thatsaid lipoproteins remain substantially unchanged.
 2. A pretreatmentmethod according to claim 1, wherein said enzyme is cholesterol oxidaseor cholesterol dehydrogenase.
 3. A method for quantitating cholesterolexisting in a specific lipoprotein in a sample, which comprises causingcholesterol dehydrogenase, which acts upon free cholesterol as asubstrate, to act upon said sample with said lipoprotein containedtherein to convert only said free cholesterol under conditions that saidlipoproteins remain substantially unchanged; and then measuring saidcholesterol, which exists in said specific lipoprotein, by using asubstance which acts upon said specific lipoprotein only.
 4. Aquantitation method according to claim 3, wherein said specificlipoprotein is high density lipoprotein.
 5. A method for quantitatingcholesterol existing in a specific lipoprotein in a sample, whichcomprises causing an enzyme, which acts upon free cholesterol as asubstrate, and a reaction accelerator, which is selected from theflufenamic acid, mefenamic acid, 2,2′,6′,2″-terpyridine, tiglic acid,fusidic acid, betamethasone acetate, monesin and mevinlin, to act uponsaid sample with said lipoprotein contained therein to convert only saidfree cholesterol under conditions that said lipoproteins remainsubstantially unchanged; and then measuring said cholesterol, whichexists in said specific lipoprotein, by using a substance which actsupon said specific lipoprotein only.
 6. A quantitation method accordingto claim 5, wherein said enzyme is cholesterol oxidase and/orcholesterol dehydrogenase.
 7. A quantitation method according to claim5, wherein said specific lipoprotein is high density lipoprotein.
 8. Amethod for quantitating free cholesterol in a sample which containsvarious lipoproteins, which comprises conducting a cholesterol oxidasereaction in said sample in the presence of a reaction acceleratorselected from flufenamic acid, mefenamic acid, 2,2′,6′,2″-terpyridine,tiglic acid, fusidic acid, betamethasone acetate, monensin andmevinolin, and then measuring said free cholesterol.